pharmaceutics Article Preparation of Electrospun Small Intestinal Submucosa/Poly(caprolactone-co-Lactide-co-glycolide) Nanofiber Sheet as a Potential Drug Carrier Nguyen Thi Thu Thao †, Surha Lee †, Gi Ru Shin †, Youngji Kang, Sangdun Choi and Moon Suk Kim * Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea; [email protected] (N.T.T.T.); [email protected] (S.L.); [email protected] (G.R.S.); [email protected] (Y.K.); [email protected] (S.C.) * Correspondence: [email protected]; Tel.: +82-31-219-2608; Fax: +82-31-219-3931 † These authors contributed equally to this work. Abstract: In this work, we chose small intestine submucosa (SIS) as a drug carrier because SIS possesses good biocompatibility, non-immunogenic property and bio-resorbability, and performed electrospinning for preparation of nanofiber sheets (NS). For the preparation of drug-loaded elec- trospun SIS nanofiber sheets as a drug carrier, we used poly("-caprolactone-ran-L-lactide) (PCLA) copolymers to improve the electrospinning performance of SIS. The electrospinning of SIS and PCLA provided the electrospun SIS/PCLA (S/P)-nanofiber sheet (S/P-NS) with adjustable thickness and ar- eas. The electrospun S/P-NS showed different porosities, pore sizes, diameters and tensile strengths depending on the ratios between SIS and PCLA. The electrospun S/P-NS was used as a drug carrier of the dexamethasone (Dex) and silver sulfadiazine (AgS) drug related to anti-inflammation. Dex- loaded S/P-NS and AgS-loaded S/P-NS was successfully fabricated by the electrospinning. In the in vitro and in vivo release, we successfully confirmed the possibility for the sustained release of Dex Citation: Thao, N.T.T.; Lee, S.; Shin, and AgS from the Dex-S/P-NS and AgS-S/P-NS for three weeks. In addition, the sustained Dex and G.R.; Kang, Y.; Choi, S.; Kim, M.S. AgS release suppressed the macrophage infiltration. Collectively, we achieved feasible development Preparation of Electrospun Small of SIS nanofiber sheets for a sustained Dex and AgS delivery system. Intestinal Submucosa/Poly (caprolactone-co-Lactide-co-glycolide) Keywords: electrospinning; nanofiber sheets; small intestine submucosa; drug; sustained delivery Nanofiber Sheet as a Potential Drug Carrier. Pharmaceutics 2021, 13, 253. https://doi.org/10.3390/ pharmaceutics13020253 1. Introduction Academic Editor: Janusz Kasperczyk Small intestine submucosa (SIS) derived from the submucosal intestinal layer is an Received: 19 January 2021 acellular, collagen-based extracellular matrix (ECM) with over 90% collagen content [1]. For Accepted: 9 February 2021 several decades, SIS has been commonly harvested from pigs and is one of the most studied Published: 11 February 2021 biomaterials in many biomedical applications [2–4]. SIS is an ideal biomaterial because of its good biocompatibility, non-immunogenic property, and bio-resorbability. Based Publisher’s Note: MDPI stays neu- on these key characteristics, some biomedical products (see Cook Biotech, Vetrix BioSIS, tral with regard to jurisdictional clai- etc.) using SIS have been developed and commercialized [5]. Additionally, SIS has been ms in published maps and institutio- approved by the Food and Drug Administration (FDA) for several clinical products [5,6]. nal affiliations. Recently, our research group and others have investigated SIS sheets, sponges, and hydrogels for biomedical applications [7–9]. Although SIS is an excellent candidate for biomedical applications, the utilization of SIS as a drug carrier is limited. Nanofiber sheets (NS) can be used in various biomedical applications such as drug Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. carriers, wound healing, scaffolding for tissue engineering, etc. [10,11]. Over the past This article is an open access article few years, the development of NS for biomedical applications has grown remarkably. distributed under the terms and con- Among several fabrication techniques for NS, electrospinning is one of the simplest and ditions of the Creative Commons At- cost-effective methods [12–17]. Successful NS electrospinning depends on the appropriate tribution (CC BY) license (https:// selection of solvents and polymers. creativecommons.org/licenses/by/ Various synthetic and natural materials can be used to fabricate electrospun NS [18,19]. 4.0/). Among the several options for materials, it was expected that electrospinning SIS would Pharmaceutics 2021, 13, 253. https://doi.org/10.3390/pharmaceutics13020253 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 253 2 of 14 generate electrospun SIS nanofiber sheets (SIS-NS). However, electrospinning of SIS demon- strates the limitations of common electrospinning solvents (e.g., 1,1,1,3,3,3-hexafluoro-2- propanol) for controlling the physical properties of electrospun SIS-NS, etc. On the other hand, synthetic polyesters such as poly(lactic acid) (PLA), poly(glycolide) (PGA), poly(D,L-lactic-co-glycolic acid) (PLGA), and poly("-caprolactone) are widely used for electrospinning because they make electrospinning relatively easy, and they can be used to easily adjust the properties of the electrospun polyesters [20–22]. Therefore, we hypothesized that an electrospinning solvent that is commonly used for synthetic polyesters could be applied to the electrospinning of SIS and synthetic polyesters to improve the physical properties of electrospun SIS-NS. Among several syn- thetic polyesters, poly("-caprolactone-ran-L-lactide) (PCLA) was selected due to several potential advantages such as the ease of preparation and controllable biodegradability and biocompatibility in vivo [23,24]. Thus, in the first part of this study, electrospinning was performed with the addition of various concentrations of PCLA into SIS in order to improve the electrospinning perfor- mance of SIS and to modulate the physical properties of the electrospun SIS/PCLA (S/P) nanofiber sheet (S/P-NS). Dexamethasone 21-phosphate disodium salt (Dex), a type of corticosteroid medication, is used to treat several skin diseases, inflammatory and auto-immune disorders, and rheumatic disorders as well as for adjunctive therapy for short-term administration [25]. Silver sulfadiazine (AgS) is a topical antibiotic used on partial-thickness and full-thickness burns to prevent infection [26]. The structural differences between Dex and AgS cause different solubility in water. Dex is easy to dissolve in water and therefore suitable for rapid administration and repeated administration, as required [27]. AgS is less water-soluble, and thus is only suitable for very limited administration [28]. To expand the utilization of Dex or AgS, a novel delivery system is needed to enable the sustained administration of Dex or AgS. In light of this, the second aim of this study was to prepare Dex-loaded SIS/PCLA (S/P) nanofiber sheets (Dex-S/P-NS) and AgS- loaded SIS/PCLA (S/P) nanofiber sheets (AgS-S/P-NS) for a sustained Dex and AgS delivery system. Dex-S/P-NS and AgS-S/P-NS would provide a means of maintaining concentrations of Dex and AgS within their implanted positions for a defined period under physiological conditions, as well as evaluating the biocompatibility of the Dex-S/P-NS and AgS-S/P-NS over a defined period (Figure1). More information on these research topics will enable the development of suitable nanofiber sheets for a sustained Dex and AgS delivery system. Pharmaceutics 2021, 13, 253x FOR PEER REVIEW 3 of 14 Figure 1. Schematics of the experimental procedures for (a) preparation ofof drug-loadeddrug-loaded smallsmall intestineintestine submucosasubmucosa (SIS)/(SIS)/ poly(ε-caprolactone-ran-L-lactide) (PCLA) (S/P) solution, (b) preparation of electrospun drug-loaded SP nanofiber sheet poly("-caprolactone-ran-L-lactide) (PCLA) (S/P) solution, (b) preparation of electrospun drug-loaded SP nanofiber sheet (NS), and (c) in vivo evaluation of drug-loaded SP-NS. (NS), and (c) in vivo evaluation of drug-loaded SP-NS. 2. Materials and Methods 2. Materials and Methods 2.1.2.1. Materials Materials TheThe initiator initiator methoxy methoxy poly(ethyleneglycol) poly(ethyleneglycol) (MPEG, (MPEG, average average molecular molecular weight weight Mn = 750 g/mol) and the catalyst Sn(Oct)2 were purchased from Sigma Aldrich (St. Louis, MO, Mn = 750 g/mol) and the catalyst Sn(Oct)2 were purchased from Sigma Aldrich (St. Louis, USA)MO, USA)and used and usedwithout without further further purification. purification. Lactide Lactide (LA; (LA;Boehringer Boehringer Ingelheim, Ingelheim, Blanque- Blan- fort,quefort, France) France) was was recrystallized recrystallized twice twice in inethyl ethyl acetate, acetate, and and ε-caprolactone"-caprolactone (CL; (CL; Aldrich, Aldrich, 2 Milwaukee,Milwaukee, WI,WI, USA) USA) was was distilled distilled over over CaH CaH2 under under reduced reduced pressure. pressure. 1,1,1,3,3,3-Hexafluoro- 1,1,1,3,3,3-Hex- afluoro-2-propanol2-propanol (HFIP, >99%) (HFIP, was >99%) obtained was obtained from Fluka from (Sigma-Aldrich, Fluka (Sigma-Aldrich, MO, USA) MO, as USA) an elec- as antrospinning electrospinning solvent. solvent. Collagenase Collagenase from Clostridium from Clostridium histolyticum histolyticum (C7657, >1200 (C7657, CDU/mg >1200) CDU/mg)was purchased was frompurchased Sigma from Aldrich. Sigma Dexamethasone Aldrich. Dexamethasone 21-phosphate 21-phosphate disodium salt disodium (Dex) and saltsilver-sulfadiazine (Dex) and silver-sulfadiazine (AgS) were purchased (AgS) were from purchased Sigma-Aldrich from (St Sigma-Aldrich Louis, MO, USA). (St Louis, Other MO,materials USA). were Other used materials without were further used purification. without further purification. 2.2.2.2. Preparation Preparation of of Small Small In Intestinaltestinal Submucosa Submucosa Powder NativeNative SIS SIS sheets were prepared using prev previouslyiously reported methods [[3].3]. The native sheetssheets were were then then crushed crushed into into powder powder by by a a freezer freezer mill mill (6700, (6700, SPEX; SPEX; Metuchen, Metuchen, NJ, NJ, USA) USA) inin liquid liquid nitrogen. nitrogen. The The SIS SIS powder powder was was treated treated in in 0.1% 0.1% pepsin and 3% acetic acid for 48 h andand then neutralized by 1 M NaOH solution.